SummaryIn Arabidopsis thaliana, several MYB and basic helix-loop-helix (BHLH) proteins form ternary complexes with TTG1 (WD-Repeats) and regulate the transcription of genes involved in anthocyanin and proanthocyanidin (PA) biosynthesis. Similar MYB-BHLH-WDR (MBW) complexes control epidermal patterning and cell fates. A family of small MYB proteins (R3-MYB) has been shown to play an important role in the regulation of epidermal cell fates, acting as inhibitors of the MBW complexes. However, so far none of these small MYB proteins have been demonstrated to regulate flavonoid biosynthesis. The genetic and molecular analyses presented here demonstrated that Arabidopsis MYBL2, which encodes a R3-MYB-related protein, is involved in the regulation of flavonoid biosynthesis. The loss of MYBL2 activity in the seedlings of two independent T-DNA insertion mutants led to a dramatic increase in the accumulation of anthocyanin. In addition, overexpression of MYBL2 in seeds inhibited the biosynthesis of PAs. These changes in flavonoid content correlate well with the increased level of mRNA of several structural and regulatory anthocyanin biosynthesis genes. Interestingly, transient expression analyses in A. thaliana cells suggested that MYBL2 interacts with MBW complexes in planta and directly modulates the expression of flavonoid target genes. These results are fully consistent with the molecular interaction of MYBL2 with BHLH proteins observed in yeast. Finally, MYBL2 expression studies, including its inhibition by light-induced stress, allowed us to hypothesise a physiological role for MYBL2. Taken together, these results bring new insights into the transcriptional regulation of flavonoid biosynthesis and provide new clues and tools for further investigation of its developmental and environmental regulation.Keywords: flavonoid, transcription, network, MYB, bHLH, TTG1. IntroductionFlavonoids are secondary metabolites that fulfil important biological functions and provide useful metabolic and genetic models for plant research, including the analysis of transcriptional regulation of gene expression (Koes et al., 2005;Lepiniec et al., 2006;Peer and Murphy, 2007;Taylor and Grotewold, 2005;Winkel-Shirley, 2001). Flavonoids are involved in protection against various biotic and abiotic stresses, they play roles in the regulation of plant reproduction and development and act as signalling molecules with the biotic environment. Besides these physiological functions, there is a growing interest in these secondary metabolites due to their potential benefits for human health (Halliwell, 2007; Luceri et al., 2007); therefore, improving our understanding of the regulation of flavonoid biosynthesis is an important objective.Although structural genes can be efficiently targeted for crop improvement, the use of regulatory genes seems to be at least as promising (Bovy et al., 2007; Grotewold et al., 940 ª 2008 The Authors Journal compilation ª 2008 Blackwell Publishing LtdThe Plant Journal (2008Journal ( ) 55, 940-953 doi: 10.1111Journal...

MicroRNAs (miRNAs) regulate gene expression posttranscriptionally through RNA silencing, a mechanism conserved in eukaryotes. Prevailing models entail most animal miRNAs affecting gene expression by blocking mRNA translation and most plant miRNAs, triggering mRNA cleavage. Here, using polysome fractionation in Arabidopsis thaliana, we found that a portion of mature miRNAs and ARGONAUTE1 (AGO1) is associated with polysomes, likely through their mRNA target. We observed enhanced accumulation of several distinct miRNA targets at both the mRNA and protein levels in an ago1 hypomorphic mutant. By contrast, translational repression, but not cleavage, persisted in transgenic plants expressing the slicing-inhibitor 2b protein from Cucumber mosaic virus. In agreement, we found that the polysome association of miR168 was lost in ago1 but maintained in 2b plants, indicating that translational repression is correlated with the presence of miRNAs and AGO1 in polysomes. This work provides direct biochemical evidence for a translational component in the plant miRNA pathway.

Background: Phosphorylation of eIF2α provides a key mechanism for down-regulating protein synthesis in response to nutrient starvation or stresses in mammalian and yeast cells. However, this process has not been well characterized in plants